Sargassum thunbergii. After consecutive UV-B radiation treatments, we used metabonomics models to analyze and compare the maximum photosynthetic electron transport rate (ETRmax), central carbon cycle metabolism (CCCM) gene expression level, CCCM enzymic activities [pyruvate dehydrogenase and citrate synthase (PDH and CS)], and carbon-based metabolite  content in male and female S. thunbergii. The results showed that under low and high UV-B radiation, the ETRmax values and six targeted CCCM gene expression levels were significantly higher in males than in females. Under high UV-B radiation, only the CS activity was significantly higher in males than in females. There was no significant difference in PDH activity between males and females. The CCCM models constructed using the metabonomics analysis demonstrate that S. thunbergii males and females exhibit obvious gender differences in their responses to UV-B radiation, providing us with a new understanding of the macroalgal gender differences under UV-B radiation, as past investigations always underestimated their diecious characteristics.The enhancement of ultraviolet-B radiation (UV-B) radiation reaching the Earth’s surface due to ozone layer depletion is an important topic. Macroalgal species growing in the intertidal zone are often directly exposed to UV-B radiation periodically as the tide changes. In order to better understand the response of macroalgae to UV-B stressed condition, we studied the dominant dioecious intertidal macroalgae  Sargassum thunbergii  is a brown intertidal dioecious seaweed that forms seaweed beds along the northwestern Pacific coast. Seaweed beds have high economic and ecological value  in 2021. The laboratory culture of S. thunbergii followed the procedures in our previous studies . The results of our daily monitoring showed that during the reproductive stage of S. thunbergii (June–August), the average UV-B radiation intensity in Qingdao from 10:00 to 14:00 was 2.29 W·m−2·s−1, and the maximum instantaneous intensity could reach 3.7 W·m−2·s−1. From 6:00 to 18:00 the average UV-B radiation intensity was no more than 1.2 W·m−2·s−1. According to these data, the high and low UV-B radiation levels in our experiments were set to 2.5 and 1 W·m−2·s−1, respectively. PAR was set as the normal illumination (control group); PAR with 1 W·m−2·s−1 UV-B radiation was the low intensity group; and PAR with 2.5 W·m−2·s−1 UV-B radiation was the high intensity group. The radiation treatment duration was 12 h per day. There were six replicates of each experimental group. After 3 days of treatment, 80 mg of thalli samples was quickly weighed, ground, and stored in liquid nitrogen at −80°C. The samples underwent metabonomics analysis using liquid chromatography-mass spectrometry (LC/MS) equipped with an electrospray ionization (ESI) source. The samples were separated with an Agilent 1290 Infinity LC ultra-high performance liquid chromatography system (UHPLC) and a hydrophilic interaction liquid chromatography column (HILIC). They were then analyzed with a Triple TOF 5600 mass spectrometer . The data were subjected to one-way ANOVA to evaluate differences in the metabolites among treatments, with a significance level of p < 0.05.After 10 days of habituated culturing, healthy S. thunbergii macroalgae under different amounts of UV-B radiation. At the beginning of the experiment, the S. thunbergii genders were determined and labeled before PAM analysis was carried out. All treatments included PAR/+UV-B radiation exposure (8 h duration) at day 0, 1, 3, and 5.A pulse amplitude-modulated (PAM) fluorometer  was used to measure the rapid light curves (RLCs) of male and female 2 into glyceraldehyde 3-phosphate (G3P). This process connects carbon fixation with the Calvin cycle and directly affects photosynthetic rates. In the next step, fructose-1,6-bisphosphatase (FBP), citrate synthase (CS), acetyl-CoA carboxylase (ACC), succinic dehydrogenase (SDH), and sorbitol dehydrogenase (SODH) co-worked in CCCM, which were connecting to carbon assimilation and metabolism processes, and as rate-limiting enzymes affect the synthesis and accumulation of subsequent metabolites. Although pyruvate phosphodikinase (PPDK) can dissipate excess light energy in photoautotrophic organisms, it cannot complete the inorganic carbon assimilation process, including the synthesis and extension of carbon skeletons  and One-Step. A gDNA Removal and PrimeScript™ RT reagent Kit with gDNA Eraser  was used for reverse transcription . After total RNA extraction, agarose gel electrophoresis (1% agarose w/v) and a Picodrop spectrophotometer  were used to determine RNA quality and concentrations. Each sample was analyzed three times. The 18S rRNA and β-actin were selected as reference genes. The genes’ relative expression patterns were analyzed using the 2-ΔΔCt method. The results were presented as target gene fold changes in the treated group divided by fold changes in the control group.In the first step of CCCM, ribulose-l,5-bisphosphate carboxylase/oxygenase (Rubisco) is catalyzed by COkeletons . The expS. thunbergii have been determined in previous research (After 3 days of treatment with PAR (+UV-B), 500 mg of macroalgae was collected and ground for metabolite content and enzymatic activity determination. Appropriate kits A081-1-1  and spectrophotometric methods were used to determine the amount pyruvic acid, which was based on its reaction with 2,4-dinitrophenylhydrazine to form pyruvic acid-2,4-dinitrophenylhydrazine appearing red in alkaline solution. The amount of pyruvic acid in the sample can be calculated by measuring the absorbance of the sample at 520 nm. Soluble sugar, total amino acids, and lipids are the key carbon skeleton metabolites participating in macroalgal physiological activity and stress resistance . The amoresearch  and theiAppropriate kits (BC0385 and BC1065)  were used to test the activity of pyruvate dehydrogenase (PDH) and citrate synthase (CS), respectively. PDH activity detection is based on the dehydrogenation of pyruvate and 2,6-dichlorophenol indigo, detected with 605 nm light absorption. The activity was detected based on the difference before and after the reaction. CS catalyzes acetyl-CoA and oxaloacetic acid to produce citrate co-enzyme A, which is further hydrolyzed to produce citric acid. This reaction causes DTNB to transform into yellow TNB with absorbance at 412 nm.t-tests. Statistical significance was considered at the p ≤ 0.05 level. Pearson’s correlation analysis was used to analyze the relationships among ETRmax, gene expression levels , enzymic activities (PDH and CS) and the metabolites content  based on the results of the 3-day experiment in dioecious S. thunbergii. Statistical significance was evaluated at the p ≤ 0.05 level.Fisher’s exact test was used to perform KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway enrichment analysis and calculate the significance level of metabolite enrichment. The metabolomic data analysis details and parameter settings can be found in previously published research . The phyS. thunbergii. Thirty-three metabolites were significantly regulated under different treatments in female S. thunbergii. Precisely 64 species of significantly regulated metabolites  are present in amino acids, sugar, and nucleotides. These compounds were derived from the carbon fixation process and distributed by the CCCM. In the CCCM model  in male and female S. thunbergii under different treatments. Before UV-B radiation treatments, there were no significant differences in ETRmax between males and females . After 5 days, males had significantly higher amounts than females under low and high UV-B radiation. Although extended time or high intensity UV-B radiation decreased the ETRmax values, male S. thunbergii always had more stable potential photosynthetic capacity than females.rubisco, fbp, ppdk, cs, sudh, acetyl-CoA, and sodh) based on RT-qPCR analysis. The results are shown as the expressed quantities of target genes in the treatment group compared to the expressed quantities in the corresponding control group.fbp had the lowest expression levels in females, significantly lower (84.98%) than that in the control group. The greatest change in male macroalgae occurred with the cs gene (6.57 times that in the control group). In female macroalgae, the greatest change occurred with the sodh gene (17.68 times that in the control group). Low intensity UV-B radiation did not dramatically promote the expression of sodh genes in male algae  than control group).Under low-intensity UV-B radiation, the expression of seven targeted genes was not inhibited in male macroalgae (their expression was higher or the same as in the control group). The rubisco, ppdk, and fbp were obviously suppressed in female macroalgae. However, they maintained normal transcript levels in males as compared to the control group. With the high-intensity UV-B radiation treatment, the sodh gene in female macroalgae had the highest value (21.65 times the controls).Under high-intensity UV-B radiation, the carbon fixation related genes S. thunbergii is shown in p>0.05), 21.76% (p>0.05), and 21.72% (p>0.05) higher in males than in females respectively. With respect to individual females or males, there was no significant difference in PDH activity in the two UV-B radiation treatments.The enzymatic activity of PDH and CS in male and female −1  and from 12.22 to 34.55 U·g−1  in the three treatment groups.CS activity in the control group and the low-intensity UV-B radiation treatment group showed no significant differences between male and female macroalgae. Low-intensity UV-B radiation could stimulate the activity of CS, which was increased by 170.74% and 86.97%, respectively, in males and females compared to the control group. Under high-intensity UV-B radiation, the CS activity was significantly (1.03 times) higher in males than in females. The CS activity in females was similar to that of the control group. CS activity ranged from 14.81 to 27.68 U·gS. thunbergii after different treatments were examined after 3 days of UV-B irradiation. There were no significant differences between the amount of pyruvate in male and female S. thunbergii in the three experimental groups  than in females . In contrast, there was a significant difference between male and female macroalgae in the amount of lipids. The amount was higher in females  than in males. In the low-intensity UV-B treatment group, the biosynthesis of soluble sugar and total amino acids in females were enhanced as compared to the control group. There were no significant differences in soluble sugar and total amino acid contents. The macroalgal soluble sugar and lipid contents in the females were higher  than those in the males. The amount of total amino acids was 11.41% lower in females than in males (p>0.05). The high-intensity UV-B treatment significantly altered the distribution of all targeted materials between male and female S. thunbergii. The lipid content was more than two times higher in females than in males. The amount of sugar and amino acids was significantly higher in males than in females.Proportion and regulation in soluble sugar, total amino acid, and lipid in male and female S. thunbergii.Metabolomic models, photophysiological RLCs, gene expression patterns, enzymatic activity, and the levels of carbon-based metabolites were analyzed to understand changes in CCCM pathway regulated mechanisms and their carbon-based metabolites under UV-B stimulation as well as differences in the responses of male and female S. thunbergii under no stress. Leucine can be used as a source of dissolved organic carbon. Its uptake has been verified to be lower than that of other amino acids  and nitrogen (N) metabolism related pathways. Several N-based metabolites constitute the “nitrogen pool” in macroalgae, balancing nitrate reduction, ammonium assimilation, photosynthesis, and protein synthesis  could be guaranteed, especially in male and female S. thunbergii stressed by high UV-B radiation. In previous studies, macroalgal photosynthetic capacities and their carbon fixation processes showed direct correlations with marine carbon assimilation so they received more attention than other topics  remained slightly increased under low-intensity UV-B radiation, but under high-intensity UV-B radiation, it increased by 5.08 times compared to the control group. Under high-intensity UV-B radiation, D-sorbitol was 2.43 times higher in males than in females. This transcriptional regulation influenced the carbon-based metabolites, which also means that in male S. thunbergii, D-sorbitol biosynthesis served as an important process for adapting to UV-B stress. In addition, D-sorbitol reacts as the key metabolite in plant stress resistance  showed that soluble sugars and amino acids served as the regulated metabolites in male S. thunbergii responding to UV-B radiation. The lipid accumulation was a key regulatory process in the response of females to UV-B radiation stress. Overall, these results provide deeper insights into the environmental stress tolerance mechanisms of male and female S. thunbergii, which is helpful for evaluating gender differences in the response of the brown macroalgae to UV-B radiation in the intertidal zone.Parameters involved in photophysiological ETRThe original contributions presented in the study are included in the article/YS conceived and designed the experiments, performed the experiments, analyzed the data, contributed reagents/materials/analysis tools, prepared figures and/or tables, authored or reviewed drafts of the paper, and approved the final draft. YZ and SS performed the experiments, analyzed the data, contributed reagents/materials/analysis tools, prepared figures and/or tables, authored or reviewed drafts of the paper, and approved the final draft. JC and JW analyzed the data, contributed reagents/materials/analysis tools, authored or reviewed drafts of the paper, and approved the final draft. QL and XT conceived and designed the experiments, analyzed the data, contributed reagents/materials/analysis tools, authored or reviewed drafts of the paper, and approved the final draft. All authors contributed to the article and approved the submitted version.This research was funded by the NSFC-Shandong Joint Fund (U1806213), National Natural Science Foundation of China (42176154) and the National Key R&D Program of China (2019YFD0901204).www.letpub.com) for its linguistic assistance during the preparation of this manuscript.We thank LetPub (The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Objective: We aim to establish a deep learning model called multimodal ultrasound fusion network (MUF-Net) based on gray-scale and contrast-enhanced ultrasound (CEUS) images for classifying benign and malignant solid renal tumors automatically and to compare the model’s performance with the assessments by radiologists with different levels of experience.Methods: A retrospective study included the CEUS videos of 181 patients with solid renal tumors  from June 2012 to June 2021. A total of 9794 B-mode and CEUS-mode images were cropped from the CEUS videos. The MUF-Net was proposed to combine gray-scale and CEUS images to differentiate benign and malignant solid renal tumors. In this network, two independent branches were designed to extract features from each of the two modalities, and the features were fused using adaptive weights. Finally, the network output a classification score based on the fused features. The model’s performance was evaluated using five-fold cross-validation and compared with the assessments of the two groups of radiologists with different levels of experience.Results: For the discrimination between benign and malignant solid renal tumors, the junior radiologist group, senior radiologist group, and MUF-Net achieved accuracy of 70.6%, 75.7%, and 80.0%, sensitivity of 89.3%, 95.9%, and 80.4%, specificity of 58.7%, 62.9%, and 79.1%, and area under the receiver operating characteristic curve of 0.740 : 0.70–0.75), 0.794 (95% CI: 0.72–0.83), and 0.877 (95% CI: 0.83–0.93), respectively.Conclusion: The MUF-Net model can accurately classify benign and malignant solid renal tumors and achieve better performance than senior radiologists.Key points: The CEUS video data contain the entire tumor microcirculation perfusion characteristics. The proposed MUF-Net based on B-mode and CEUS-mode images can accurately distinguish between benign and malignant solid renal tumors with an area under the receiver operating characteristic curve of 0.877, which surpasses senior radiologists’ assessments by a large margin. Nowadays, cancer remains a serious threat to human health worldwide. The incidence of renal cancer is increasing annually, with more than 400,000 new cases every year worldwide . Clear cDeep learning has shown promising results in the classification and diagnosis of renal tumors over the past few years , which dIn this study, we aim to establish a multimodal fusion deep neural network based on gray-scale ultrasound and CEUS images to discriminate between benign and malignant solid renal tumors. The performance of the multimodal fusion model is compared with that of the models built on single-modal data, as well as junior and senior radiologists’ assessments.This retrospective study was approved by our joint institutional review boards, and anonymized data was shared through a data-sharing agreement between institutions  (No. 18PJ149 and No. 20SXQT0140). Individual consent for this retrospective analysis was waived. From June 2012 to June 2021, the information for 1547 cases was obtained from the surgical pathology database in the Pathology Department of The Second Clinical Medical College of Jinan University and The Affiliated Nanchong Central Hospital of North Sichuan Medical College.The inclusion criteria were as follows: (1) preoperative CEUS examinations were performed before surgery, and (2) all cases were confirmed by pathological diagnosis after surgery. Patients were excluded based on the following criteria: (1) renal pelvis cancer and other rare types of renal malignancies; (2) patients did not receive the ultrasound and CEUS examinations; (3) poor image quality, and (4) pathologic stage ≥ T2b. CEUS examinations were performed using the following three ultrasound systems: LOGIQ E9 , Resona7 , and IU22 , with a 1.0–5.0 MHz convex probe. CEUS was carried out by ultrasound machines with contrast-specific software and a bolus of 1.0∼1.2 ml microbubble contrast agent  via an antecubital vein followed by 5.0 ml normal saline with a peripheral 18∼22 G needle. The CEUS digital video was at least 3∼5 min long each time. During contrast-enhanced imaging, low-acoustic power modes were used with a mechanical index of 0.05∼0.11. All the CEUS examination videos were retrospectively analyzed by two groups of radiologists with different levels of experience (three junior radiologists with more than 5∼6 years of experience in CEUS imaging and three senior radiologists with more than 10∼15 years of experience in CEUS imaging).In this study, we used the following phase terms: (1) cortical phase began 10∼15 s after injection, and (2) medullary phase approximately began 30∼45 s after injection until the microbubble echoes disappeared. The entire course of CEUS was saved as Digital Imaging and Communication in Medicine format.CEUS videos were annotated using the Pair annotation software package . In eachThe dataset used in this study contained B-mode and CEUS-mode images, and they were in one-to-one correspondence. Therefore, we proposed the MUF-Net to take full advantage of the multimodal features, which can independently extract features from each of the two modalities and learn adaptive weights to fuse features for each sample.The overall architecture of MUF-Net is shown in Notably, to improve the feature learning ability of each backbone, we added two classifiers All experiments were conducted using five-fold cross-validation. For data splitting, we ensured that the images from the same patient went into either the training set or the test set to avoid the data leakage problem. To avoid model overfitting, data augmentation techniques were applied to the training set, which included random spatial transformations, random non-rigid body transformations, and random noise.The dataset used in this study had a weak class imbalance problem. The ratio of benign images to malignant images was 3:5. Re-sampling techniques  were popAll backbones used in these experiments were pretrained on ImageNet. All models used in the experiments were implemented by PyTorch on a NVIDIA 3090 GPU. The stochastic gradient descent optimizer was used with a learning rate of 0.05 which was halved every 10 epochs. In each round of five-fold cross-validation, models based on B-mode, CEUS-mode, and B + CEUS mode were trained for 100 epochs, respectively, and the models with the highest accuracy on the validation set were saved.Original uncropped CEUS videos and images were evaluated by three junior and three senior radiologists and manually classified as benign or malignant. The radiologists were blinded to any clinical information of the patients. Intraclass correlation coefficients (ICCs) were used to evaluate the inter-rater agreement within each radiologist group, with an ICC greater than 0.75 indicating good agreement.p value < 0.05 was considered statistically significant.All statistical analyses were performed using the SciPy package in Python (version 3.8). Depending on whether data conformed to a normal distribution, continuous variables were compared using the Student’s t-test or the Mann-Whitney U test. The non-ordered categorical variables were compared by the chi-square test. Receiver operating characteristic (ROC) curve analysis was used to evaluate the performance of junior radiologists, senior radiologists, individual modality-based networks, and MUF-Net. In addition, we also used other metrics to evaluate model performance from various aspects, including sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV). Comparison of the difference between areas under the ROC curve (AUCs) was performed using the Delong test. A two-sided p = 0.017). Regarding gender distribution, there was a significant difference between these two groups , with male patients being more frequent in the malignant group than in the benign group. Patient characteristics are shown in The age of the patients in the benign tumor group was less than that of the patients in the malignant tumor group (58.36 ± 14.06 vs. 53.31 ± 14.00 years) .The AUC, accuracy, sensitivity, specificity, PPV, and NPV of EffecientNet-b3 network trained on B-mode images were 0.820 (95% CI: 0.72–0.83), 74.5%, 75.0%, 77.0%, 73.4%, and 62.3%, respectively . The AUCThis study explored the performance of deep learning based on ultrasound images for benign/malignant classification of solid renal tumors. We proposed the MUF-Net for fusing complementary features of two modalities, which used two independent EffecientNet-b3 as backbones to extract features from B-mode and CEUS-mode ultrasound images. Our method reached expert-level diagnostic performance and had a higher diagnostic PPV compared with radiologists.CEUS is an important supplement to conventional ultrasound, CT, and MRI in diagnosing solid renal tumors. Compared with conventional ultrasound, CEUS can display perfusion characteristics of renal tumors in cortical and medullary phases in real-time, which is an important tool for improving differential diagnosis of benign and malignant renal tumors. In this study, we observed that the deep learning models built on either B-mode or CEUS-mode images achieved better performance than junior or senior radiologists. Moreover, the deep learning model combing B-mode and CEUS-mode images further improved the classification performance. This indicates that B-mode and CEUS-mode images have complementary information for diagnosing solid renal tumors. To verify this point, we used class activation maps  to visuaAccording to the results from the comparative experiments, we found that the performance was similar between the two models built on B-mode or CEUS-mode images. The B-mode images-based model had slightly better performance, which might be due to the different microcirculatory perfusion characteristics of solid renal tumors. Solid renal tumors of different histopathological types have different vascular density, fat content, blood flow velocity, and the severity of arteriovenous fistulas.Lin et al. reported an AUC of 0.846 for the classification of benign and malignant renal tumors on enhanced CT images using inception-v3 . Xu et aThis study had several limitations. First, the classical and well-established convolutional neural network, EffecientNet-b3, was selected as the backbone based on previous experiments, which may not be optimal. The characteristics of multimodal ultrasound imaging data need to be analyzed in-depth, and other deep neural networks will be attempted in the future to see if better performance can be achieved. Second, only images of tumor regions were cropped and used in data analyses, and regions of tumor periphery might be able to provide more information to improve model performance, which requires further experimental analyses. Third, our model was implemented using the ultrasound images collected from two hospitals only. A larger dataset acquired from more hospitals with different types or models of ultrasound equipment may have the potential to further improve the performance and generalization ability of our model.


Nearly 55 percent of patients are said to be affected by the neurological effects of COVID-19. COVID-19 was shown to be related with stroke in 0.9 to 5% of people. It's critical to assess the impact of COVID-19 on the outcomes of acute ischemic stroke. The goal of this study was to look at the outcomes and characteristics of patients who had an acute ischemic stroke due to covid-19 infection.P = 0.12). There was a high in-hospital mortality rate among patients with COVID-19 with a rate of 11.7% compared to 4.04% among the control group (P = 0.02). At discharge, the number of patients with mRS > 2 was higher (P = 0.001) among the COVID-19. There was a correlation between the mean levels of D-Dimer , the severity of COVID-19 , and mRS > 2. The participants in this study were 399 people who had had a stroke. COVID-19 positivity was confirmed in 77 cases, while COVID-19 negativity was confirmed in 322. In the COVID-19 and control groups, the average age of the patients was 65.4 ± 10.2 and 65.3 ± 11.8, respectively. The Covid-19 and control groups had a mean stroke onset of 5.2 ± 2.1 and 5.7 ± 3.8 h, respectively (. This includes elderly patients, patients with severe COVID-19, patients with high levels of D-Dimer, and those with high NIHSS.Despite receiving equal acute care as non-COVID-19 patients, COVID-19 patients had more severe strokes and had worse outcomes. This includes a high chance of death while in the hospital as well as a significant level of disability. Neurologists should use timely and effective therapies, particularly for patients who are at a higher risk of having a stroke COVID-19 is a novel strain of the -coronavirus family that is enclosed, positive-sense, single-stranded RNA virus. COVID-19 has become a prominent source of illness and mortality worldwide since its emergence in December 2019. The WHO labelled the disease a pandemic in March 2020 . AccordiThe respiratory system is most affected by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Extrapulmonary symptoms like cardiac failure, renal insufficiency, and neurological problems affect a considerable proportion of SARS-CoV-2 patients. Nearly 55 percent of patients are said to be affected by the neurological effects of COVID-19. COVID-19 was shown to be related with stroke in 0.9 to 5% of people –10. ThisStroke is linked to a higher rate of mortality in the hospital. Furthermore, it has a significant negative impact on the patient's life and is linked to disability, psychological distress, and a lack of social interactions , 15. It These limitations result in controversial results, making it difficult to draw firm conclusions from the available evidence. Therefore, the current study aimed to assess the outcome and characteristics of patients with acute ischemic stroke associated with covid-19 infection.This prospective cohort multicentric study involved 399 patients presented with acute stroke patients diagnosed according to WHO criteria . PatientAfter obtaining approval from the local ethics committee, patients who agreed to participate gave their signed informed consent after explanation of the trial benefits and hazards. All procedures were carried out in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Declaration of Helsinki and its later amendments or comparable ethical standards.The patients were then divided into two groups. Within four days of admission, the COVID-19 group had a positive nasal SARS-CoV-2 PCR test. Patients who had clinical characteristics indicative of COVID-19 at the time of admission and were determined to be SARS-CoV-2 positive at any point during the first ten days of hospitalization were also included in the study. Patients who were consistently SARS-CoV-2 negative or who were never tested because they did not develop symptoms or evidence of COVID-19 made up the other group. The study excluded patients with traumatic intracerebral hemorrhage, subarachnoid hemorrhage, subdural hematoma, and extradural hematoma., which varies from 0 (no symptoms) to 6 (death) [Demographic data, risk factors, stroke characteristics, management, and stroke severity  were recorded. For ischaemic strokes. The TOAST  classification was either taken from the discharge summary or was inferred from the clinical team’s documented assessment of likely stroke aetiology [ (death) .P-value < 0.05. Statistical analysis was performed using SPSS software version 25 for Windows , [The student's T-test was used to compare continuous normally distributed data in the form of mean and standard deviation (SD). The median and range were used to describe non-normally distributed data, and the Mann–Whitney U test was used to compare between the case and control groups. Categorical variables were reported as a number or a percentage, and groups were compared using Pearson's chi-square test and Fisher's exact test. For non-parametric variables, Spearman's rank correlation coefficient was used to do correlation analysis. The binary logistic regression model was used to determine factors linked to stroke outcomes in COVID-19 patients. The significance is established when two-sides P < 0.001), platelet count (P < 0.001), and C-reactive protein (CRP) ((P < 0.001), in comparison to the control group. There was a statistically significant low mean of total lymphocytic count among COVID-19 group, in comparison to the control group with a mean of 0.86 ± 0.12 and 1.33 ± 0.28, respectively (P < 0.0001). There was no difference between age, gender, and comorbidities between both groups , and 322 patients were SARS-CoV-2 negative (control group), while 18 patients were excluded from the study. The mean age of the included patients was 65.4 ± 10.2 and 65.3 ± 11.8 in the COVID-19 and control groups, respectively. There were 45 (58.4%) males in the COVID-19 group and 195 (60.6%) males in the control group. Patients with COVID-19 had higher mean levels of total leucocytic count (TLC) (P = 0.12). On admission, 42 (54.5%) patients were treated with antiplatelets among the Covid-19 group, relative to 174 (54%) patients within the control group (P = 0.94). Furthermore, 12(15.6%) and 59(18.3%) patients received Alteplase among the Covid-19 and control groups (P = 0.62), respectively. The most frequently affected territory was the anterior circulation, affecting 46 (59.74%) and 218 (67.7%) patients within the Covid-19 and the control groups (P = 0.52). The cause of the stroke was large vessel occlusion in 21(27.3%) patients within the Covid-19 group and 68(21.1%) patients within the control group (P = 0.69). although percentage of patients with fits in Covid-19 cases was higher than control group yet P value was non-significant (0.07) . Within the COVID-19 and control groups, the median NIHSS was 7 (0–42) and 12 (1–42), respectively. Patients with COVID-19 had a higher in-hospital death rate of 11.7 percent compared to 4.04 percent in the control group (P = 0.02). The most common cause of death in the Covid-19 group was respiratory failure and pulmonary embolism, accounting for 4 (5.19%) and 3 (3.89%) patients, respectively. At discharge, the number of patients with mRS > 2 was statistically substantially greater (P = 0.001) in the COVID-19 group, with 50 (64.93%) patients compared to 133 (41.3%) patients in the control group . There was an association between mean D-Dimer levels , COVID-19 severity , NIHSS , and mRS > 2 in this regard. Neither the NIHSS (P = 0.966) nor the severity of COVID-19 (P = 0.978) were significant predictors of mRS > 2 at discharge in the binary logistic regression model (Tables As for Predictors of stroke outcomes in COVID-19 Patients, the age of the patients and mRS > 2 had a positive connection (The present COVID-19 situation has highlighted the importance of determining the pandemic's impact on healthcare . While sCOVID-19 patients experienced poor stroke outcomes, according to the current study. In patients with COVID-19, the in-hospital death rate was around three times that of the control group. At patients discharge, most COVID-19 patients had poor stroke outcomes, with a risk of roughly 1.5 times that of COVID-19 patients. Furthermore, patients who were elderly, had severe COVID-19, had high levels of D-Dimer, or had a high NIHSS had a higher chance of bad stroke outcomes at discharge. In the binary regression model, however, neither of these characteristics was significantly linked with mRS > 2. COVID-19 has been linked to the onset of ischemic stroke via thrombotic and inflammatory processes , 24.The development of a cytokine storm and activation of the innate immune system, embolic events propagated by pre-existing or new-onset arrhythmias, and hypoxia-induced ischemia secondary to severe pulmonary illness are among the primary hypothesized processes. These conditions exacerbate the effects of stroke, resulting in additional neurological impairments and, as a result, poor outcomes. These findings matched those of Mart-Fàbregas and colleagues, published in 2021. They discovered that patients who had an ischemic stroke linked to COVID-19 had a more severe stroke. In comparison to patients without COVID-19, they reported a 3.1-fold increase in mortality risk within 72 h of admission . The sigThis finding can help epidemiologists and doctors treating stroke on the front lines, especially in places where COVID-19 prevalence is high . COVID-1According to the findings of the current study, some stroke victims avoided obtaining emergency care in hospitals for fear of infection. This explains why there are fewer hospitalized stroke patients and a higher NIHSS score, which indicates a severe stroke. Patients having mild strokes during the COVID phase may be less likely to seek medical care , 28. RouWhen compared to earlier findings on stroke in COVID-19 patients, the new study had several advantages. It was based on a multicenter hospital registry to begin with. COVID-19 patients' outcomes were compared to non-COVID-19 patients who had an acute stroke and were treated with the same management procedures. These protocols should be tailored to the new in-hospital pathways created to prevent pandemic viral spread. Other investigations, like as chest CT, had an impact on stroke care time metrics in all patients admitted during the pandemic. The current study, on the other hand, has some drawbacks. To begin with, a variety of circumstances could have influenced whether patients sought emergency medical attention, were admitted to a hospital, and received extensive treatment. Some COVID-19 and ischemic stroke patients may have died before reaching the hospital, while others with lesser symptoms or treatment limits may have remained at home. This could have resulted in an underestimation of the total rate of ischemic stroke in COVID-19 patients. In addition, data from a registry set up to detect cardiac and thromboembolic problems in COVID-19 patients was used in part of our retrospective investigation.Despite receiving equal acute care as non-COVID-19 patients, COVID-19 patients had more severe strokes and had worse outcomes. This includes a high chance of death while in the hospital as well as a significant level of disability. Neurologists should use timely and effective therapies, particularly for patients who are at a higher risk of having a stroke. This includes elderly patients, patients with severe COVID-19, patients with high levels of D-Dimer, and those with high NIHSS. Further studies with adequate sample size and a more extended follow-up period are required to tackle the current study's limitations.


Amomum tsao-ko is well-known as a spice as well as a Chinese traditional herb. This study aimed to identify the bioactive constituents in the powder of methanol extract from Amomum tsao-ko (PMEAT) and to evaluate the hypoglycemic and antioxidant effects of PMEAT, in vitro and in vivo. We identified 36 phytochemicals in PMEAT by employing HPLC-MS/MS. PMEAT solution was found to have potent α-glucosidase-inhibiting activity  in vitro, twice as strong as that of acarbose . To investigate the hypoglycemic activity of PMEAT in vivo, we studied the impact of low-dose PMEAT (the addition of 100 mg/kg PMEAT to the mice diet) and high-dose PMEAT (200 mg/kg PMEAT addition) treatments in STZ-induced diabetic mice. After 6 weeks of intervention, significantly decreased fasting blood glucose (FBG) (p < 0.05), significantly decreased area under the curve (AUC) of the oral glucose tolerance test (p < 0.05), significantly decreased HOMA-IR (p < 0.05), and significantly increased HOMA-β (p < 0.05) were observed in the high-dose PMEAT group. Moreover, we performed an antioxidant activity experiment in vitro. The results showed that PMEAT had a strong ability to scavenge DPPH  as well as ABTS free radicals . In an animal experiment conducted on oxidative damage mice model which was induced by D-glucose and a high-fat diet, we observed significantly increased dismutase (SOD) (p < 0.01), glutathione (GSH) (p < 0.01), and glutathione peroxidase (GSH-Px) (p < 0.01) and significantly reduced malondialdehyde (MDA) and 8-ISO-prostaglandin-PGF2α (8-ISO-PGF2α), after treatment with PMEAT for 90 days. In conclusion, this study reveals the therapeutic potential of Amomum tsao-ko for the treatment of diabetes and helps us discover new antioxidant candidates from natural sources.The dried fruit of  In the last three decades, especially high-calorie diets have been shown to affect lipid metabolism and lead to fat accumulation, resulting in obesity, which is a cause of various chronic diseases such as diabetes, hypertension, and cardiovascular disease . Many biAmomum tsao-ko Crevost et Lemaire (A. tsao-ko), a perennial plant classified into the Zingiberaceae family, is a medicinal herb discovered in Southeast Asia. In China, it is mainly distributed in the Guangxi, Guizhou, and Yunnan provinces. A. tsao-ko can not only be used as an edible spice but also has therapeutic effects on stomach disorders, dyspepsia, nausea, abdominal pain, and throat infections, as recorded in the Chinese Pharmacopoeia. Flavonoids, coumarins, phenols, and diterpenoids were found as bioactive constituents in A. tsao-ko extracts, which have been reported to have the activity of anti-obesity  to study their physiological regulating activities , only A. tsao-ko exhibited hypoglycemic prosperity. Based on the above studies, the activity of the residue, acetone extract, chloroform extract, and methanol extract of A. tsao-ko were, respectively, assessed by performing animal experiments , in vivo and in vitro. This study may provide important evidence for the utilization and pharmaceutical development of methanol extracts of A. tsao-ko.The present study was conducted as a further analysis to identify the bioactive constituents contained in the methanol extracts of α-Glucosidase Detection Kit was purchased from Shanghai Guyan Industrial Co., Ltd. . Acarbose standard sample was purchased from Shijiazhuang Huarong Pharmaceutical Co., Ltd. . The Insulin ELISA kit was purchased from Shijiazhuang Huiduan Biotechnology Co., Ltd. . Superoxide dismutase (SOD) assay kit, reduced glutathione (GSH) assay kit, glutathione peroxidase (GSH-Px) assay kit, malondialdehyde (MDA) assay kit, and mouse 8-iso prostaglandin F2α (8-ISO-PGF2α) ELISA kit were purchased from Nanjing Jiancheng Bioengineering Institute . Other reagents were all analytical grade.A. tsao-ko fruits were purchased from Kunming herbal market in Yunnan province, China. We prepared PMEAT according to the methods of Yan et al. with slight modifications  and sieved (100 mesh). A total of 50 g fruit powder was added to the mixed alcohol extracting solution  and stayed still for 12 h. After being mixed for 5 min , the mixed liquor was crushed for 10 min  with an ultrasonic cell pulverizer . After being shrunk and filtered by vacuum, the crushed mixture was concentrated and turned into a filter cake, which was added with a half amount of the mixed ethanol extract  to complete the first-time extraction. Next, extraction was repeated twice in the same way and the filtrate was collected from three extractions. A 60 g activated silica gel powder was then added to the filtrate and the powdery mixture was obtained after being concentrated using a rotary evaporator . The powder sample was then put in a self-made silica gel hydrogen column  and eluted successively with 600 mL chloroform, 600 mL acetone, and 600 mL methanol. Thus, we obtained the methanol eluent, which was then filtered by a microporous membrane (0.22 μm). The powder of methanol extract of A. tsao-ko was finally obtained after the evaporative concentration and freeze-drying.Dried ications . First, Chromatographic separation was performed in a Thermo Ultimate 3000LC system equipped with a Zorbax Eclipse C18 column (1.8 μm × 2.1 × 100 mm) maintained at 30°C . The mob2CO3 solution (pH 6.8). PBS was taken as blank control and was used as a background solution instead of α-glucosidase solution to eliminate the influence of the ground effect. The optical density (OD) of each well was detected using a microplate reader at a wavelength of 405 nm. The inhibition rate against α-glucosidase of two test solutions was calculated as follows:The determination procedure of α-glucosidase (α-GIA) inhibiting activity was referred to as the method of Chen et al. . In brieEight-week-old male ICR mice  [Animal license number: SCXK(Jing)2016-0006] were obtained from Beijing Vital River Laboratory Animal Technology Co., Ltd . Animals were housed in a controlled environment  with free access to pellet diet and water. The mice were used for experiments after 1 week of acclimatization.n = 10), fed the basic mouse fodder , and a high-fat diet group (n = 40), fed high-fat diet . From the 7th week, the mice in the HFD group were injected intraperitoneally with 150 mg/kg·BW STZ (Sterptozotocin) solution (STZ was dissolved with 0.1 mol/L pH 4.5 citrate buffer) after fasting for 12 h. Seventy-two hours after injection and fasting for 12 h again, tail vein blood was taken to determine the fasting blood glucose (FBG) concentration of the mice. The mice in the HFD group with HBG levels ≥ 11.1 mmol/L were selected as diabetic mice  as follows: the model group (Mod), the low-dose PMEAT group (PMEAT-L), and the high-dose PMEAT group (PMEAT-H). From week 8 to week 13, the control group was fed with basic mouse fodder, the model group was fed with high-fat diet, the PMEAT-L group was fed with high-fat diet adding 100 mg/kg PMEAT, and the PMEAT-H group was fed with high-fat diet adding 200 mg/kg PMEAT .The oral glucose tolerance tests were performed in week 8 (before the PMEAT intervention) and week 13 (after the PMEAT intervention) on every group. After 10 h of fasting, all mice were intraperitoneally injected with a glucose solution (1.5 g/kg·BW). The blood glucose levels of the tail tip were measured at 0 (baseline), 15, 30, 60, and 120 min after the injection, using a one-touch glucometer . The integrated glucose response  over 2 h after the glucose overload was calculated as follows , the blood of mice in four groups was collected from the retro-ocular venous after fasting for 10 h. The fasting blood glucose was measured with the OneTouch UltraTM rapid blood glucose meter. The remaining blood was centrifuged  to collect plasma, and plasma insulin was quantified by an enzyme-linked immunosorbent assay (ELISA) kit. Homeostatic model assessment for insulin resistance (HOMA-IR) and homeostatic model assessment for β cell function (HOMA-β) were calculated based on fasting blood glucose (FBG) and fasting insulin level . Calcula50 value (mg/mL).The DPPH assay was performed according to the method of Cui et al. with slight modifications . In brie50 value (mg/mL).The ABTS assay was undertaken by the method of Cui et al. with slight modifications . First, 3 at a ratio of 10:1:1 (v/v/v). Subsequently, 0.1 mL of the diluted test sample and 3 mL FRAP reagent were added to the test tube and reacted at 37°C in the dark for 5 min. The absorbance was measured at 596 nm. With the standard solution of FeSO4 as the reference substance, the FRAP result of the samples was presented as the millimoles of FeSO4 required to achieve the same absorbance.The FRAP assay was performed using the method of Guo et al. . The FRAin vivo, we performed another 90-day animal experiment based on the oxidative damage mouse model (n = 12), a model group , a low-dose PMEAT group , and a high-dose PMEAT group . The mice in the control group were fed a basic diet for 8 weeks. The mice in the model group were fed a high-fat diet (HFD) for 8 weeks. The mice in the PMEAT-L and PMEAT-H groups were treated with HFD adding 100 mg/kg PMEAT and HFD adding 200 mg/kg PMEAT for 8 weeks, respectively. During this same 8-week period, the mice in each group except the control group were intraperitoneally injected with D-galucose (125 mg/kg·d) once per day , then blood and liver tissues were collected after sacrificing , 26. Blot-test were performed to examine the differences among treatment groups for the analysis of the hypoglycemic indicators  and antioxidant indices . P-value < 0.05 indicated that the difference between groups was statistically significant.Statistical analysis was performed using SPSS version 26.0. Data were tested for normal distribution with the Shapiro–Wilk test. One-way ANOVA followed by Tukey's-SHD and Student's To elucidate the substance basis of the pharmacologic effect, a HPLC-MS/MS full-spectrum analysis in positive and negative ionization modes was performed . The fraA. tsao-ko may be mainly attributed to this category of phytochemicals.Based on the content data of the constituents and the findings, we inferred that phenols, flavonoids, and oligosaccharides were probably the main compounds that exhibited the hypoglycemic and antioxidant activities , 28. Amoin vitro hypoglycemic effect of PMEAT, α-GIA was investigated (50 0.273 mg/mL), PMEAT had a higher α-GIA (IC50 0.154 mg/mL). When the concentration of the PMEAT solution was 0.5 mg/mL, the inhibition rate reached 63.72%. The above strong inhibitory activity against α-glucosidase was probably attributed to a variety of phenolic and coumarins compounds in PMEAT. Many researchers have also confirmed that 4-Hydroxycoumarin, Avicularin, Epicatechin, Hesperetin, Quercetin, Procyanidin B1, and Quercetin-3β-D-glucoside have varying degrees of α-glucosidase inhabitation activities (To assess the stigated . We obsetivities –51.p < 0.05). No significant difference was observed in PMEAT-L group when compared with the Mod group. As shown in p < 0.05). The HOMA-IR of the PMEAT-H group was significantly (p < 0.05) lower than that in the Mod group as shown in p < 0.05) higher than that in the Mod group presented in in vivo anti-diabetic properties. The compounds probably prevented oligosaccharides in food from decomposing into monosaccharides, and thus reducing the glucose absorption in the intestine and protected islet cells, and consequently enhanced glucose tolerance . As shown in A. tsao-ko is inferior to rutin and curcumin in total antioxidant capacity.In this article, various assays  were used to comprehensively evaluate the antioxidant activities of in vitro antioxidant activity. As explicated in a review article by Chaitanya et al.  and aromatic rings in polyphenols has been reported to enhance hydrophobic (π-π) interactions with α-glucosidase . FlavonoA. tsao-ko was a complex mixture and could not exhibit obvious antioxidant capacity at low concentrations. The antioxidant capacity increased considerably when the PMEAT solution reached a higher active concentration. This is consistent with the results of our previous studies .LY designed and supervised the study. LX, DY, YL, HJ, JC, and LH were involved in animal experimentation. YL and HJ performed the statistical analyses and interpreted the data. LX and DY drafted the manuscript. JC revised the article critically for important intellectual content. All authors approved the final version of the manuscript to be submitted. All authors contributed to the article and approved the submitted version.This study was funded by the Foundation of the Department of Education of Hebei Province (QN2020525) and the Medical Science Research Project, Health Commission of Hebei Province .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


To explore the latent pathogenesis of circRNAs in osteoarthritis (OA), as well as their function mechanism.  The murine chondrocytes with and without OA were involved and used for in-depth sequencing. Herein, we carried out subsequent bioinformatics analysis to disclose the expression pattern, characteristics of circRNAs based on gene ontology, and the KEGG pathway analyses. Then sequencing data were used to deduce the interaction between circRNA and miRNA. The potential miRNA response elements for the annotated circRNAs and relevant target genes were forecasted on the basis of TargetScan and miRanda. For chondrocytes, the effect of the overexpression of the screened circRNA for apoptosis was spotted by flow cytometry as well as Western Blot.  466 diverse circRNAs in the 23,787 spotted circRNAs were both significantly and differentially transcribed. CircKMT2E was upregulated more than two folds in chondrocytes with OA compared with normal tissues, exhibiting an expression trend opposite to miR-140-5p. We disclosed that circKMT2E could possess mutual effect with miR-140-5p by way of AGO proteins. Thus, circKMT2E was verified to have functioned as a molecular sponge targeting miR-140-5p. Therefore, circKMT2E may be at work in the pathogenesis of OA. Further, the sponge connection between circKMT2E and miR-140-5p was proved on the basis of a dual-luciferase reporter assay. Besides, miR-140-5p was speculated can bind TLR4 by bioinformatics analysis. Further PCR analysis found the relative expression level of TLR4, caspase-3, and Bax in the OA groups presented significant upregulation. Overexpression of circKMT2E can promote apoptosis of chondrocytes.  The upregulation of circKMT2E is involved in the chondrocyte apoptosis of the pathogenesis of OA through activation of TLR4 by the sponge function of miR-140-5p. Osteoarthritis (OA) as a sort of degenerative disease that occurs in joint that causes decreased quality of life or even physical disability, is mainly depicted by the gradual inflammatory undermine of articular cartilage , 2. The Since chondrocytes are the single cells that exist in articular cartilage, chondrocytes play a role in the progression of pathology of OA, especially by relying on both cytokine production and apoptosis. , 5 ChondmiR-140 as a kind of chondrocyte-specific miRNAs, is indispensable for the proliferation of endochondral bone. If miR-140 becomes deficient, osteal deformity as well as dwarfism would be given rise to . The miRThe in-depth transcriptomic data of both healthy and OA murine cartilaginous tissues were downloaded from the NCBI bioproject database with accession numbers PRJNA798112 and PRJNA817158, in which, after stratified sampling, three healthy samples and three OA samples were enrolled in this research. Wild male C57BL/6 mice were used for the experimental procedures when aged 8 weeks old. The destabilisation of the medial meniscus incised the right knee of the mice with a patella tendon in the middle, and the tendon between the medial meniscus and the tibial plateau was cut in virtue of a diminutive surgical scissors. For the control group, only arthrotomy was implemented, namely medial meniscotibial ligament cutting was exempted.t-test. The p value was corrected using the Benjamini & Hochberg method. [p value ≤0.05.Sequence reads with high quality were then matched to the reference transcriptome using the STAR software (v2.5.1b) . CircRNA method.  The diffp value was computed on the basis of Fisher's exact test .The enrichment analyses on the basis of GO as well as KEGG that aims to disclose differentially expressed circRNAs were implemented. GO is a methodic and organized database in order to depict both genes and their products. It not only covered molecular function but also revealed biological process as well as cell component. With the help of the pathway analysis from KEGG, the signaling pathways that containing circRNAs as well as their biological functions can be inferred. The relevant t-test was applied on the basis of GraphPad Prism 8.0 to compute the significance for differences.The abundance of circRNAs was calculated by Ballgown and computed by FPKM. The threshold value of FPKM in each group was 0.5, which means the circRNA would be deemed as expressed in this group if FPKM >0.5. For circRNAs expression, Student's https://www.bioinf.com.cn/) and the UCSC genome browser were used to annotate the structure of circKMT2E and its positions on parental genes, respectively. The CircMir1.0 software, on the basis of the miRanda 2010 deliver (https://www.microrna.org/microrna/getDownloads.do) and RNAhybrid-2.1.2, was applied to annotate putative bundling situations of miR-140-5p on circKMT2E transcripts.The underlying miRNA reaction elements for the annotated circRNAs and target gene were predicted using the custom-written software which on the basis of both TargetScan and miRanda . CircPrimer1.2 (6) on the basis of Lipofectamine 2000  at 37°C. Afterwards, the transfected cells, after 48 h transfection, were reaped to accomplish the following procedures.The human primary chondrocytes cells  were cultured in Primary Chondrocyte Culture System . CircKMT2E mimics and circRNA control were synthesized by Aksomics Inc. . The aforementioned circRNAs (20 nM) were transfected into the human primary chondrocytes cells . After 48 hours, we spotted the fluorescence activities on the basis of the Luc-Screen Assay System , and computed the comparative fluorescence activities.−ΔΔCT method. Beta-actin was used as an internal reference. Based on the GraphPad Prism 8.0, we used Student's t-test to ensure the significant differences for circRNAs.Total RNAs were extracted on the basis of TRIzol . Whereafter, the density of extracted RNAs was assessed based on NanoDrop , as well as the value of OD260/OD280 within 1.8–2.1. Then, cDNAs were compounded on the basis of the SuperScript Kit . Further qRT-PCR was carried out on the ViiA 7 Real-time PCR System  on the basis of the PowerTrack SYBR Green Master Mix . Relevant primers were engineered by primer 5.0 on the basis of the longitudinal transcripts . The relqRT-PCR was applied to check the expression extent of apoptosis-related gene, circRNAs with differential expression, as well as miR-23b-3p. Involved primer sequences: beta-actin: 5′-GAACCGACTTCTCCCCTTGT-3′ and 5′-TCGTCTGTTAGGTGGATGCTT-3′. circKMT2E: 5′-TGACCTCCTTCGCCACTTAC-3′ and 5′-CATAACAGCGTCACCACAGC-3′. miR-140-5p: 5′-AAGAGACTGGGGTGTGGAAA-3′ and 5′-TCTGAAGTTTTCCATTTCTCTGC-3′. TLR4: 5′-GGGATCACATTGCCAGGGAT-3′ and 5′-CAGTGCGTGTCGTGGAGT-3′. Caspase-3: 5′-CTCGCTTCGGCAGCACA-3′ and 5′-AACGCTTCACGAATTTGCGT-3′. Bax: 5′-CAAAGGGAATACATTGCCAGA-3′ and 5′-TGTGGCCAACTGCTGAAA-3′.μg) went through electrophoresis on the basis of 10% SDS-PAGE, therewith, transferred proteins onto PVDF membranes. Then membranes were blocked at room temperature for 2 h on the basis of 5% bovine serum albumin . Then, all samples were soaked by primary antibodies all night at 4°C and then soaked by HRP-conjugated secondary antibodies at 26°C for 2 h. Eventually, the enzyme-linked chemiluminescence kit  and CD-touch  were applied to photograph the blots which can be quantified on the basis of Image Lab software. Above-mentioned antibodies were purchased from Abcam.Total protein, with the help of RIPA buffer including protease inhibitor, was extracted. To explore the protein concentrations, the BCA protein assay kit  was applied in this study. Proteins . Then, we used a computational program on the basis of the Ensembl transcriptome GTF file as well as the DCC software to ascertain circRNAs.Based on unique junction reads ≥123,787, diverse circRNAs were detected across all samples Figures . For thep < 0.05 for the purpose to seek circRNAs that differentially expressed  as an example and predicted target mRNAs of the five leading miRNAs binding to circKMT2E using TargetScan and miRanda . We annoin vitro, we used human primary chondrocytes cell, respectively, transfected by circKMT2E and circRNA-control to detect apoptosis rate using flow cytometry and apoptosis-related protein using Western blot. As flow cytometry results disclosed that the upregulation of circKMT2E significantly enhanced the apoptosis rate of chondrocytes cells, while no significant differences were found between the circRNA-control group and the blank control group (To verify the promoting function for apoptosis of circKMT2E ol group . In addiSynovium, as one of the most crucial components of the joint system, is formed by loose connective tissue. The core functions of the synovium include weakening joint surface friction, secreting synovial fluid, offering nutrients to articular cartilage, restraining joint adhesion, restraining cell fragments in the joint cavity, and assimilating miscellaneous metabolites. When stress of injuries, such as trauma, degeneration, infection, and rheumatism, comes to the joint, the partly undesirable inflammatory reaction of the synovium will be stimulated to take part in the purpose of repairing the injury to the articular cartilage. It is found that OA synovitis usually presents chronic as well as low-grade inflammation as the dominating feature, which may intimately come to the pathogenetic process of the cartilage damage of OA. Accordingly, endeavoring to adjust the condition and circumstances of synovitis in order to rebuild the local microenvironment of the OA joint as well as accelerate the repair progress of chondrocytes is promising to serve as a viable approach for the clinical treatment of OA. Whereas, the formation progress and molecular mechanism of OA synovitis remains unclear.β-induced apoptosis as well as inhibit OA. Ding et al. disclosed that miR-93 can inhibit cellular apoptosis of chondrocytes as well as inflammation occurs in OA on the basis of targeting the TLR4/NF-κB signaling pathway.Previously, plenty of researches have disclosed that, during the process of OA, synovial tissue was motivated to generate inflammatory responses and ultimately to generate and contribute to the constitution of chronic synovitis by the cellular fragments from lifeless chondrocytes and degradative cartilage matrix. . TherefoIn this study, we first disclosed the expression patterns of circRNAs in murine chondrocytes related to OA. In murine chondrocytes with OA, circKMT2E was disclosed to be the most significant upregulated circRNA. Further study revealed circKMT2E possesses a negative correlation with miR-140-5p, which is one of the chondrocyte-specific miRNAs and is indispensable for normal endochondral bone proliferation in mice. PCR also verified the enlarged expression of circKMT2E and diminished expression of miR-140-5p in OA tissue.κB pathway. [Inflammation-induced catabolism includes cell dedifferentiation, matrix metalloproteinase reaction, restraint the expression of type II collagen as well as aggrecan core protein are firmly dominated by TLR-dependent immune reaction. , 15 For pathway. , 20.Therefore, the relative expression levels of TLR4, caspase-3, and Bax in the normal and OA groups were detected by PCR. TLR4 and these two apoptosis-related proteins all presented significant upregulation in the OA group. To moreover check on the circKMT2E/miR-140-5p/TLR4 regulatory pathway, our research team performed a dual-luciferase reporter assessment to confirm the combinative connection between circKMT2E and miR-140-5p, they used transfected cells to verify the ability of circKMT2E to promote apoptosis. Wang et al.  previousThis study has a few limitations. First, the visualized results of the overexpression study were not carried out owing to our limited laboratory conditions. Second, the specific signaling pathway of chondrocyte apoptosis were not further explored. Third, the murine gene knock-out research were not involved.In this present study, differentially expressed circRNAs in murine chondrocytes with OA were analyzed. We preliminarily disclosed the sponge interaction between miR-140-5p and OA related circKMT2E. The upregulation of circKMT2E may be involved in the chondrocyte apoptosis of OA through activating TLR4 by sponging miR-140-5p.


The DEmiRNA target genes were identified using RNAhybrid and miRanda, and only those identified by both were considered. The combined effects of the DEmiRNAs were determined using KEGG pathway analysis. Another 40 POTS and 20 normal patients were used as validation subjects. Plasma H2S was determined with a sulfide electrode, and flow-mediated vasodilation (FMD) was performed with a color Doppler ultrasound system. miRNAs were analyzed using qRT-PCR.High-throughput sequencing was used to obtain whole-blood miRNA expression profiles for 20 POTS sufferers and 20 normal children.The thresholds for defining differentially expressed miRNAs (DEmiRNAs) were an adjusted DESeq P values of 0.01 and 0.05 were used. The target gene of hsa-miR-21-5p was SP1 when the P-value is <0.01. DEmiRNAs were significantly enriched in 36 pathways (P < 0.05), in which PI3K/Akt signaling was closely related to vascular function. In the validation subjects, the plasma H2S and FMD were higher in the POTS sufferers (P < 0.05).Totally, 13 DEmiRNAs were identified through high-throughput sequencing. In the 60-member validation group, the 13 miRNAs were verified again, and it turned out that miR-21 was significantly elevated and could diagnose POTS with a 100% specificity and 92.5% sensitivity. Overall, 198 and 481 genes, respectively, were shown to be targeted by the 13 DEmiRNAs when 2S observed in POTS sufferers.Elevated whole-blood miR-21 levels serve as an indicator for POTS and may explain the increased plasma H As an orthostatic intolerance subtype, postural tachycardia syndrome (POTS) is the most frequently seen anomaly in the autonomic specialty clinics, which affects around 0.5–3 million U.S. residents, according to estimates  refer to 19- to 25-nucleotide-long short molecules of RNAs that are responsible for regulating target gene silencing after transcription. An individual miRNA is capable of targeting several hundreds of mRNAs and influential to multiple gene expressions, usually showing implications in the interactive pathways. miRNAs are vital for cell proliferation, differentiation, metabolism, apoptosis, development, and aging, and they are implicated in the pathophysiology of many diseases, such as cancers as well as neurologic and cardiovascular disorders .Prior to the head-up test, any possible autonomic function-affecting drug was discontinued by the children. The test was conducted in a tranquil room with adequate temperature. A Multi-Lead Physiological Monitor  was utilized for persistent surveillance of the cardiac rate and blood pressure during the test. The protocol constituted 10-min (at least) lying down and subsequent 10-min standing. In the event of a positive response within the 10-min standing, the test was terminated. Children with normal variation ranges of cardiac rate and blood pressure during the test underwent a head-up tilt test on the next day.−1, or if the maximum heart rate reaches the diagnostic criteria ; at the same time, blood pressure changes a little  and any two of the following symptoms present during tilting: dizziness/vertigo, thoracic tightness, headache, cardiopalmus, pallor, blurring of vision, weariness, or syncope. Any child with a positive response was diagnosed with POTS.Prior to the head-up tilt test, all children were instructed to fast for 4 h (at least) and discontinue any possible autonomic function-affecting drug. A Multi-Lead Physiological Monitor  was utilized for persistent surveillance of cardiac rates and blood pressures of these children, who were laid on a HUT-821 tilt table . Tilting of the table was performed at a 60° angle upon the cardiac rate stabilization. The surveillance of the cardiac rate and blood pressure continued until the emergence of a positive response during the first 10 min. A response would be considered positive if the cardiac rate was elevated by ≥40 beats·minFor POTS, its diagnostic criteria were (1) a normal cardiac rate in supine position; (2) over two clinical symptoms on standing, including dizziness/vertigo, aching head, lightheadedness, weariness, pallor, blurring of vision, thoracic tightness, cardiopalmus, trembling hands, and syncope; (3) cardiac rate elevation by ≥40 times/min and (or) the maximum heart rate reaches the diagnostic criteria , and at the same time, blood pressure changes a little ; (4) relief or mitigation of symptoms by recumbence and ≥1-month persistence of symptoms; and (5) exclusion of other cardiovascular, metabolic, or neurologic lesions  were drawn at 8:00 am from subjects in supine position into PAXgene Blood RNA Tubes (BD). Immediately following blood sampling, the tubes were inverted 8–10 times mildly. In every tube, whole blood (2.5 ml) and additives (7 ml) were added. The blood samples were allowed to stand for 4 h at ambient temperature and subsequently preserved at −80°C.RNA extraction was accomplished from the Blood RNA tubes (PAXgene), followed by quality control. Central analysis was performed on all the quality control outcomes at the end of 2020. After ambient temperature thawing and agitation, the samples were all observed for identifying whether the tubes contained any blood clot. For the automatic RNA extraction from the samples, the Blood RNA kit (PAXgene) was utilized in combination with a QIA cube. Following elution with buffer, the RNA was preserved at −80°C.Regarding the miRNA library input material, 2 μg of RNA was used per sample. A Small RNA Sample Prep Kit  was utilized for the creation of miRNA sequencing libraries, and the sequences were assigned to every sample through the addition of index codes. Initially, the total RNA was ligated to a 3′ adaptor, and the resulting mixture was subjected to a 2-min incubation at 70°C for the secondary RNA structure breakage, followed by instant placement on ice. The next step was a 1-h specific ligation of the 3′ adaptor to the miRNA 3′ end by the T4 Rnl2 (T4 RNA Ligase 2) truncated at 28°C. An additional 1-h ligation of the ligated fragment to a 5′ adapter proceeded at 28°C by T4RNA ligase. Utilizing SS IV (SuperScript IV), the synthesis of the first-strand cDNA was accomplished as per the manufacturer's instructions. For the polymerase chain reaction (PCR) amplification, 5-fold Phusion HF buffer, RP1 (RNA PCR primer), RPLX (RNA PCR primer index) primer, dNTP, and Phusion DNA polymerase were utilized. Subsequently, a polyacrylamide gel (6%) was used to purify the PCR products. Thereafter, the 140- to 160-bp-long RNA fragments  were recovered and then dissolved using elution buffer (8 μL). Lastly, a Qubit spectrophotometer was utilized to assess the library concentration, while a Bioanalyzer system (Agilent 2100) and a High-Sensitivity DNA Kit were employed to examine the library quality. The index-coded samples were clustered on a cBot station with a TruSeq PE Cluster Kit v3-cBot-HS (Illumina) as per the manufacturer's instructions. Following cluster generation, sequencing of the prepared libraries proceeded on a HiSeq 2000 platform (Illumina), and single-end 50-bp reads were produced to assess transcriptomics. The sample quality control, experimentation, and data analysis were all accomplished by Genesky Biotechnologies Inc. in Shanghai, China.via the Bioconductor's edgeR. The resulting normalized profiles for all the sample replicates were subsequently analyzed by hierarchical clustering and principal component analysis (PCA) to control quality and evaluate inter-sample similarities.Exploiting the miRDeep2's quantifier module, the expression profile was generated for the known mature miRNA, which offers read counts for known miRNAs. The generated expression profiles of raw reads for all the sample replicates were normalized by the trimmed mean of the M-value approach P values. The thresholds for defining DEmiRNAs were an adjusted DESeq P of <0.05 and a log2 fold variation of ≥3.Aided by HTSeq (v0.6.1), the quantity of reads mapped to every miRNA was subjected to normalization. Then, the expected fragments per kilobase million (FPKM) number was computed for every miRNA according to the gene length and the read quantity mapped to miRNA. It is common practice to predict the levels of miRNA expression in FPKM, a value reflecting the effects of both gene length and sequencing depth for the read quantity. DESeq2's R package was exploited to analyze the DEmiRNAs. Utilizing the digital miRNA expression data, this package enables routine statistical determination of differential expression with a negative binomial distribution-based model. For the false discovery rate (FDR) control, the Benjamini–Hochberg method was employed to adjust the resulting The miRanda and RNAhybrid algorithms were used for the potential target gene recognition for the dysregulated miRNAs. We considered the miRNA target genes recognized by both algorithms.P-value and FDR. Any KEGG pathway was regarded as significant when P < 0.01.DEmiRNA joint effects on pathways were evaluated by KEGG analysis based on their associated target genes. The threshold parameters for pathway significance were −ΔΔCT method. The primers of miR-21 and U6 were as follows: 5'-TAGCTTATCAGAC TGATGTTGA-3' ; and 5'-CTCAACTGGTGTCGTGGA-3'; 5'-CTCGCTTCGGCAGCACA-3' ; 5'-AACGCTTCACGAATTTGCGT-3' .For placental isolation of total RNA in humans, an RNeasy Micro Kit  was utilized. A Nanodrop spectrophotometer  was used for the quantification of RNA. Initially, reverse transcription of total RNA (20 mg) per sample was accomplished with reverse transcriptase -involving a master mix. Real-time PCR was carried out with a PRISM 7700 Sequence Detector  as per the instructions of the manufacturer. All the Assays-on-Demand primers and probes were procured from ABI. All PCRs were run in triplicate. The reaction system contains forward primers and reverse primers  and 2 × Maxima SYBR Green qPCR Master Mix (12.5 μL), cDNA(200 ng), and nuclease-free water reaches 25 μL totally. U6 small nuclear RNA (snRNA) was used as an internal control of miR-21 levels. The expression of miR-21 was normalized to snRNA U6, and relative expression was calculated using the 22S. After washing with distilled water and drying, the electrode was soaked in the sample. Upon stabilization of readout, we documented the electrode potential. The standard curve method was employed for the computation of the H2S concentration  was collected at the cubital vein from every subject in the morning (7:00 am to 8:00 am) in a tube containing ethylenediaminetetraacetic acid and aprotinin. After mixing 0.5 mL of plasma with an equal volume of antioxidant buffer, a PXS-270 sulfide electrode  was used to determine the plasma HAn HP2500 color Doppler ultrasonographer  was utilized for the FMD test, whose transducer frequency was 7.5 MHz. The American College of Cardiology guideline was followed in this study to achieve brachial arterial assessment of the endothelium-dependent FMD , which was also used for the GO (gene ontology) and KEGG analyses. Differences were regarded as significant when P < 0.05. The datasets for this study can be found in the [ArrayExpress] [https://www.ebi.ac.uk/arrayexpress/]. The accession number is E-MTAB-10961.SPSS  was used for analyzing data. The continuous data displayed are means ± SDs, while the categorical data displayed are quantities of cases. The inter-group differences were examined by χP >0.05), while the cardiac rate varied significantly (P <0.05)  years. The 20 healthy controls also comprised 12 boys and eight girls, aged 11.2–14.1 years, with a mean of (13.1 ± 1.13) years. Insignificant inter-group differences were noted regarding age, height, blood pressure, or weight (P <0.05) .P < 0.05 are shown in We identified 135 DEmiRNAs between the two groups. Among them, 49 are upregulated and 86 are downregulated . The 32 P-value is 0.01 and 0.05, 198 and 481 genes, respectively, are shown to be targeted by the 13 miRNAs. The target gene of hsa-miR-21-5p was SP1 when the P-value is <0.01 . The target gene counts of the 13 miRNA for which a log2 fold change ≥3 is shown in is <0.01 .P-value , BP , and MF (molecular function), according to the P-value . The maiP < 0.05)  . Figure 2S and FMD are higher in POTS patients. Furthermore, the fold increase in the expression level of whole-blood miR-21 is higher in POTS patients, while miR-151b, miR-let7g, and miR-1278 were reduced (P < 0.05)  .Next, the validation participants were diagnosed by our standard criteria for defining POTS sufferers as described earlier. The levels of whole-blood miR-21 were determined by qRT-PCR. The results in 2S level in POTS patients was higher than that in the normal subjects, and the FMD of POTS patients was also increased due to the influence of plasma H2S. The increment of plasma H2S and FMD was in line with the previous study  and cystathionine γ-lyase (CSE), two enzymes relying on pyridoxal phosphate (vitamin B6)  increased the expression of miR-21, which could protect the cell from injury. Anti-miR-21 abolished the protective effects of sodium hydrosulfide by inactivating the Akt pathway. Hence, the activation of the Akt pathway regulated by miR-21 participates in the protective effects of H2S against hypoxia/reoxygenation-induced injury , and also exhibits anti-inflammatory and anti-apoptotic effects. A cell model of hypoxia/reoxygenation has suggested that incubation of sodium hydrosulfide  patients revealed that the adipokine retinol-binding protein 4 (RBP4) stimulates the PI3K/Akt pathway, which increased NO-mediated vasodilation. As a measure of the endothelium (NO)-dependent response, FMD was increased as expected  channels, which can be enhanced by NO. The associations between NO and H2S have been emphasized in the experiments in which treatment using a NO donor enhanced the endogenous production of H2S in rat aortic tissues  for relevant researchers to study. Finally, the cause of miR-21 elevation in POTS patients cannot be answered by the current study. Further studies are apparently needed to confirm our findings on miR-21 in relation to POTS and to elucidate underlying mechanisms.Despite powerful support by evidence in statistical and biological terms, there remain a few shortcomings with our study. First, although matching of the patients and the control subjects was ensured as much as possible, the sample size used for the whole-blood miRNA sequencing was small. Even though the association between miRNA and POTS was shown to be statistically significant, extra miRNA associations were probably missed since the sample size was small, and power issues were present. However, as a small sample of a pre-experimental study, 13 DEmiRNAs were identified, which was satisfactory. Second, during the validation process, it would have been advisable to ascertain the levels of plasma NO for the patients and control subjects, which would have proven our results more reliable, but corresponding evidence can be confirmed in other literature. Third, cell experiments and vascular ring tests to establish whether miR-21 mediates the expression of Hdatabase://www.ebi.ac.uk/arrayexpress/. The accession number is E-MTAB-10961. For more information, contact the corresponding author at linjing0127@xjtu.edu.cn.The raw sequencing data for this study can be found on the ArrayExpress The studies involving human participants were reviewed and approved by Xi'an Jiaotong University, school of medicine. Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin.JLin contributed to conceptualization, writing the original draft, and reviewing and editing, as well as reading, and approving the final manuscript. JS and WC assisted with conceptualization and acquisition of data and read and approved the final manuscript. JLiu contributed to conceptualization and acquisition of data and read and approved the final manuscript. LL helped with the acquisition of data and read and approved the final manuscript. FJ contributed to writing, reviewing and editing, and as well as read and approved the final manuscript. All authors contributed to the article and approved the submitted version.The work was supported by the National Natural Science Foundation of China (grant number 81803263 [to JLin]).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Subsequently, Rh moieties are covalently attached on the polymer chain with controllable grafting degree via an efficient thiol-ene click reaction. Concurrently, the effect of catalyst systems and reaction conditions on the catalytic copolymerization performance is presented, and the quantitative introduction of Rh is described in detail. Owing to its amphiphilic characteristics, the rhodamine-functionalized polyester-based block copolymer can self-assemble into micelles. With the covalent incorporation of Rh moieties, the as-formed micelles exhibit excellent absorption and fluorescence-responsive sensitivity and selectivity towards H+ in the presence of various metal cations. Moreover, the as-prepared micelles with favorable water dispersibility, good pH sensitivity and excellent biocompatibility also display appreciable cell-membrane permeability, staining ability and pH detection capability for lysosomes in living cells. This work provides a new strategy for the facile synthesis of novel biocompatible polymeric fluorescent pH nanosensors for the fluorescence imaging of lysosomal pH changes.A novel lysosome-targeting PEGylated polyester-based fluorescent pH nanosensor is fabricated by the combination of ring-opening copolymerization (ROCOP), side-group modification and subsequent self-assembly. First, a key target amphiphilic copolymer carrier for rhodamine (Rh) pH indicator is synthesized in a facile manner by the ROCOP of phthalic anhydride with allyl glycidyl ether using mPEG-OH and  Lysosome, as one of the important intracellular digestive organelles, serves as a major degradation compartment for the molecular degradation behaviors during cell metabolism ,2,3. In Although strenuous efforts have been focused on the development of polymeric fluorescent lysosomal pH nanosensors, and most of the nanosensors are efficient with high resolution for lysosomal imaging ,36,37,38Very recently, the epoxide-anhydride ring-opening copolymerization (ROCOP) catalyzed by metal-free Lewis pairs has attracted considerable attention. Due to the advantageous features of this ROCOP, such as atom-economic, enriched monomer resources and mild reaction conditions, it has become an effective and promising strategy for the synthesis of biocompatible polyesters ,43,44,45+-induced ring-opening mechanism, and have been commonly reported as an ideal choice to imaging lysosomal -2-yl)ethyl)-2 mercap-toacetamide (RhB-SH) was synthesized according to the literature procedures 0/[AIBN]0 = 1/0.3/0.1. The click reaction between the PEGylated polyester block copolymer (0.1 mmol of C=C group) and RhB-SH (0.03 mmol) were conducted under a nitrogen atmosphere. The reaction materials were first dissolved in 4 mL THF with AIBN as initiator. Then, the mixture was placed in a 70 °C oil bath and was continuously stirred for 4h. Afterwards, the reaction was stopped, the solution was filtered and the solvent was removed under vacuum. THF was used to dissolve the crude products, which were then precipitated into cold diethyl ether twice, and the final reaction products were collected and vacuum-dried at 45 °C overnight.Typically, the synthesis of rhodamine-functionalized amphiphilic copolymer was started with the ratio of reagents [C=C]A total of 50 mg of rhodamine-functionalized copolymer was first dissolved into 3 mL THF, then dropped into 25 mL deionized water, and stirred for about 1 h. Subsequently, THF was removed through dialysis against deionic water. After dilution, the aqueous solution in the dialysis bag was subjected to DLS measurement. TEM measurements were also carried out on the transmission electron microscope.https://leica-camera.com/en-int), all images were taken with an an oil immersion objective. Excitation: 560 nm, red emission collected: 560–700 nm.To evaluate the imaging of lysosomal pH changes, HeLa cells were firstly pretreated with various concentrations  of bafilomycin A1 for 9 h, then incubated with an mPEG-b-P nanosensor (5 μM) for 6 h at 37 °C. After being cultured for 24 h, each plate received 500 μL fresh medium enriched with bafilomycin A1. After incubation for 9 h, the plates were purged with fresh PBS, then incubated for additional 6 h with 5 μM nanosensor. Afterwards, fluorescence imaging was performed after HeLa cells were again washed with fresh PBS buffers (pH = 7.0) and incubated with fresh medium. On a fluorescence microscope . Channel 1 (Lyso Tracker): excitation: 488 nm, emission collected: 488–560 nm; Channel 2 (polymeric nanosensors): excitation: 560 nm, emission collected: 560–700 nm.To evaluate the imaging of co-localization, Hela cells were cultured with fresh medium containing polymeric nanosensors (50 μg/mL) for 3 h, then stained with Lyso Tracker Green DND-189 (1 μM) and incubated for 30 min. Thereafter, the cells were washed in PBS and imaged using a Zeiss 7103-channel confocal laser scanning microscopy (CLSM) . Then, the micelles were removed by washing the cells with fresh PBS repeatedly. Afterwards, 20.0 μL of MTT stock solution was added into each well and cultured for another 4h. After that, the plates were examined using a microplate reader, and 150.0 μL of DMSO was replenished. Subsequently, the system was shaken for several minutes, and the absorbance of the system was measured at 390 nm using a Bio-Rad reader . The experiments were repeated three times.The cytotoxicity of the rhodamine-functionalized polymer micelles was determined with Hela cells. Optical microscopy (Leica) was used to examine the morphology of the cells. MTT analysis was used to evaluate the cell viability of the micelles. In detail, cells were seeded in 160 μL of appropriate medium containing 10% FBS in 96-well microplates. The cells were incubated with different concentrations of micellar nanoparticles , with abundant alkenyl groups endowing the copolymer with further functionality by post-modification. The mPEG-b-P was synthesized by ROCOP of PA and AGE using mPEG-OH as the initiator and t-BuP1/Et3B as the binary catalyst . Since tn,NMR and Mn,SEC) of the corresponding PEGylated polyester indeed increased with prolonged polymerization time . After the reaction was conducted for a certain time, the viscosity of the mixture increased noticeably, suggesting the apparent molar mass of the product progressively increases, indicating the occurrence of polymerization. The copolymerization data are summarized in t-BuP1 versus BEt3 led to different copolymerization results , their SEC traces were quite different from each other; only when the feed ratio of t-BuP1 to BEt3 was 0.2:0.4, the corresponding polymer maintained an unimodal molar mass distribution  was confirmed by NMR characterization. The  spectra , entry 5 by Wang  or Yu [1 by Wang , the strn,NMR of the copolymer was calculated to be 9200. In general, the above results suggest the successful synthesis of mPEG-b-P block copolymer with a proper Mn and pendant alkenyl functional groups.Moreover, by comparing the integral of proton peak at 5.58 from AGE monomer with the proton peaks at 7.5–7.8 ppm from PA monomer, the molar ratio of PA to AGE on the copolymer backbone was calculated to be 1:1, confirming the formation of an alternating structure of the P segment. Furthermore, by comparing the integral of proton peak “a” from the residual groups of the initiator with the proton peak “g” from PA monomer A, the po1H NMR and 13C NMR spectroscopy ), a standard thiol-ene click reaction was carried out to conjugate side alkenyls with RhB-SH and to control the grafting degree of rhodamine. The RhB-SH B, which troscopy C,D. The troscopy C. Additireaction D. Moreovreaction A. Importreaction C, the grAlternatively, the percentage of rhodamine attached to the polyester backbone was also estimated by using a standard curve method. Specifically, the UV-vis absorption spectra of RhB-SH in DMF solution with different concentrations were firstly examined; then, the ratio of the characteristic absorption of the ring-closed RhB-SH at 316 nm was plotted versus the RhB-SH concentrations B and, thDh of 119 nm  in aqueous solution was investigated. Dynamic laser light scattering (DLS) analysis revealed hydrodynamic diameters f 119 nm . Concurrf 119 nm . The abohe probe A. The mihe probe B. The mPhe probe , indicatRhodamine, as one kind of classical fluorescent dyes, exhibits outstanding photophysical properties, and has been widely used as a fluorescent pH nanosensor for acidic environments. The spirolactam structure of rhodamine derivatives always exhibits sensitivity to pH variations, and can undergo a pH-dependent equilibrium between the non-fluorescent (colorless) ring-closed form and the fluorescent (colored) ring-opened form. When the situation is basic or neutral, the rhodamine remains closed, and the system is non-fluorescent; whereas the acidic situations always induce the ring-opening of spirolactam and the system exhibits strong fluorescence and a pink-to-red color  17,19,3,319,34. ex = 365 nm) are at 560 and 582 nm, respectively, which were attributed to the formation of the ring-opened rhodamine. As shown in + was also investigated  micelles as fluorescent nanosensors towards pH, and further indicates their suitability for studying acidic organelles.The responsiveness of the mPEG-b-P micelles as nanosensors towards pH was investigated by UV–vis absorption and fluorescence spectroscopies. stigated B,D. The stigated B. The ab+. The response time of the polymer micelles towards H+ was recorded systematically at a pH of 3.0  = N0 + N1eτ–t/ with different time constants  micelles are indeed sensitive and are effective nanosensors for acidic pH.Furthermore, to evaluate the fluorescent sensitivity of the mPEG-b-P micelle nanosensors to acidic pH, we also investigated the dynamic processes of fluorescence responsive behaviours of the polymer micelles induced by HH of 3.0 A. As shoonstants B. The ti−1) in tris-buffer solutions (pH 7.0) after the addition of various cations (100 μM) are shown in +, Zn2+, Ba2+, Al3+, Mn2+, Ni2+, Co2+, Cd2+, Cu2+, Hg2+, Fe3+, Mg2+, Ca2+, Pb2+, Sr2+, Cr2+ and Gd2+, in comparison with a blank sample . Whereas only when the micelle solutions were acidic, the solution emitted obviously strong fluorescence centered at 582 nm. Accordingly, the impact of cations on the absorption characteristics of the mPEG-b-P micelles agreed well with the fluorescence characteristics. As shown in 3+ was slightly stronger; however, it was only associated with the formation of small iron (III) clusters [3+. Whereas the pH value of the micelle solutions was below 6.0, the solutions showed considerable absorption characteristics, which was in accordance with the visible color change  micelles exclusively towards H+ in the presence of various cations proved that it can serve as a highly selective fluorescence nanosensor for H+ and is favorable to work as a pH indicator.The selectivity of the mPEG-b-P micelle solutions for pH was further investigated by fluorescence measurements. The representative fluorescence spectra of the copolymeric micelles  of nanosensor for 6 h at 37 °C, respectively; red fluorescence of the nanosensor within the cells was observed when the concentration of nanosensor was relatively high , demonst® Green DND-189, was selected to co-stain HeLa cells with the nanosensor , could effectively inhibit lysosomal acidification and then further induce the increase of the lysosomal pH. Taking advantage of the ability of bafilomycin A1 to adjust the lysosomal pH, we then proceeded to investigate the possibility of the mPEG-b-P nanosensor for the detection of lysosomal pH. HeLa cells were first incubated with various concentrations  of bafilomycin A1 for 9 h at 37 °C, then with 5 μM mPEG-b-P nanosensor for 6 h. With the increase of the concentration of bafilomycin A1, the fluorescence intensities within the cells obviously weakened , with abundant alkenyl functional groups was facilely synthesized by the ROCOP of PA and AGE using mPEG-OH and